Surveyor hplc system

2-DE and LC-MS/MS in human immortalized oral epithelial cells (HIOECs) and in HB96 cells were described thoroughly in our previous study [22 (link)]. Briefly, HIOECs and HB96 cells were lysed, sonicated and protein was quantified. First-dimensional IEF was completed with an IPGphor IEF System (Amersham Biosciences, Uppsala, Sweden) and second-dimensional SDS–PAGE was performed with a Hoefer SE 600 Ruby System (Amersham). Differentially expressed protein spots were excised and digested for mass spectroscopy. The peptide mixtures were isolated and identified by a Finnigan LTQ mass spectrometer coupled with the Surveyor HPLC system (Thermo, Sunnyvale, CA). Differentially expressed protein identification in MS/MS raw data was determined using the SEQUEST program in the BioWorks 3.1 software suite (University of Washington, licensed to Thermo Finnigan) based on the International Protein Index human database version 3.15.1.

The SOCS3 mimetic peptides, named KIR or KIR-ESS, were designed starting from structural and biochemical studies of SOCS3/Jak2 complex [18 (link)]. Control peptides (Ctrl1 and Ctrl2) were identical to SOCS3 KIR-ESS region with aminoacid substitutions in positions critical for SOCS3 and Jak2 interactions (Table 1). Peptides were synthesized through solid phase peptide synthesis, performed on a fully automated multichannel peptide synthesizer Syro I (Multisynthech, Witten, Germany). Preparative RP-HPLC was carried out on a Shimadzu LC-8A, equipped with a SPD-M10 AV detector and a Phenomenex C18 Jupiter column (50 × 22 mm ID; 10 μm) (Shimadzu, Japan). Peptides were then, analyzed by mass spectrometry, carried out on an LCQ DECA XP Ion Trap mass spectrometer equipped with an OPTON ESI source, operating at 4.2 kV and 320°C, and with a complete Surveyor HPLC system (ThermoFisher Scientific, Waltham, MA USA). To facilitate the peptide delivery into cell cytoplasm, the fragment 48–60 of the HIV Tat protein was conjugated to SOCS3 or to control peptides in a stepwise manner. Purified peptides were lyophilized and stored at −20°C until use.

Peptide mass spectrometry was achieved using a Surveyor HPLC system (Thermo) configured for nano flow rates using a split solvent supply coupled with an LCQ DECA XP Plus ion trap mass spectrometer (Thermo Thermo Fisher Scientific, Waltham, MA, USA). Peptides were separated with a BioBasic C18 reversed phase column (Thermo 72105–100266, Thermo Fisher Scientific) using an acetonitrile (ACN) gradient of 5 % ACN to 50 % ACN in 620 min. Flow rate was set at 500 nL per minute with 0.1 % formic acid as an ion source. Column eluate was ionized using a stock LCQ nanospray ion source operated at 2 kV applied using liquid junction just before a silica emitter. The LCQ was operated in normal scan mode with MS/MS scans of the top five most abundant ions from each precursor scan. Dynamic exclusion was enabled with a repeat count of two and a duration of two minutes. Data collection occurred over the entire 620 min gradient.

Suspensions of 0.5 g of feces were made in 1 mL of sterile demineralized water by vortexing for 3 min. Afterward, samples were centrifuged (Gusto^® High-Speed Mini Centrifuge; Heathrow Scientific LLC, Vernon Hills, IL, USA) for 8 min at 10,000 rpm. With the usage of 0.22 μm polytetrafluoroethylene (PTFE) syringe filters (Millex-GS, Merck Millipore, Darmstadt, Germany), the supernatants were filtered and subjected to high-performance liquid chromatography analysis (HPLC).
Organic acids, namely, lactate, SCFAs (acetate, propionate, butyrate, valerate), and BCFAs (isobutyrate, isovalerate) concentrations were established with the usage of the Surveyor HPLC System (Thermo Scientific, Waltham, MA, USA) equipped with the Aminex HPX-87H column of 300 × 7.8 mm in dimensions (Bio-Rad Laboratories, Hercules, CA, USA). The parameters of the analysis are presented in Table 4.
Retention times of HPLC standards were used to detect analyzed organic acids on chromatograms. Moreover, the concentration of acids was calculated with the use of the area under the established peak based on previously prepared standard curves. The results were given as micromole per gram (µmol/g).